Method and composition for hydrolyzing eggshell membrane

ABSTRACT

The present invention relates to a method for hydrolyzing eggshell membrane, comprising the step of treating a suitable amount of eggshell in a solution containing a denaturing agent, a reducing agent, a buffer, and an enzyme. The invention also relates to a composition for hydrolyzing eggshell membrane according to the preceding method.

FIELD OF THE INVENTION

The present invention generally relates to the field of obtainingsoluble protein products, and more specifically to hydrolyzing eggshellmembrane.

BACKGROUND OF THE INVENTION

Eggshell membrane is very rich in metabolites of biological interestsuch as collagen, elastin, glycosaminoglycans (hyaluronic acid,chondroitin sulfate, heparan sulfate, dermatan sulfate), as well asglucosamine. It has been published in some studies that more than 500proteins involved in the eggshell mineralization process as well asembryo protection have been identified in eggshell membrane, all of thembeing highly valuable constituents that may be used as a supplementaimed at improving skin and joint quality.

The generation of large amounts of eggshell by the industry thereforeprovides many opportunities for obtaining two fundamental raw materials:calcium carbonate (inorganic part of the shell) and membrana testaceaconsisting of two membranes, an inner membrane and another outermembrane, which are cross-linked by means of fibers forming a tight meshrendering the structure thereof insoluble.

A technical problem relating to the insoluble nature of eggshellmembrane is in fact known in the industry. Specifically, thisinsolubility is caused by the fibrous nature and the structuralcomposition of the membrane; specifically, it is largely due to thecross-linking of collagen with elastin and keratin, in addition to theabundant disulfide bonds that are part of this framework.

In the field of protein solubilization in general, proteins must bebroken down to the point where they can be integrated in the solution,something which is achieved by means of hydrolysis. Alkaline or acidichydrolysis having the drawback of breaking down part of the compositionof the original protein, reducing its nutritive value or eveneliminating constituents such that precise protein characterization isnot allowed, are generally applied. Another type of hydrolysis that iswidely used for obtaining hydrolyzed proteins is the enzymatic pathway.Many protein by-products of the industry are put to good use throughthis process, as is the case of obtaining fish hydrolysates and whey,which are used as supplements in the food industry since they maintaintheir nutritional quality.

There are therefore three fundamental ways for solubilizing proteins:acidic hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Asmentioned above, both acidic and alkaline hydrolysis have the drawbackof destroying some constituents of interest and reducing the nutritionalvalue of some proteins. Enzymatic hydrolysis has the advantage ofapplying mild conditions which allow obtaining a product with betternutritional characteristics and possible bioactivities, and is thereforepreferable. However, in the case of eggshell membrane a conventionalenzymatic hydrolysis cannot be applied due to the membrane being highlyenzyme-resistant.

Document EP 2612922 A1 discloses a method for solubilizing eggshellmembrane by means of using a protease and a reducing agent; said method,however, does not provide acceptable efficiency. Therefore, it would bedesirable to have a method for hydrolyzing eggshell membrane whichprovides improved solubilization yields.

When dealing with proteins that are extremely difficult to hydrolyze,the use of combinations of several proteinases (a cocktail) for aneffective hydrolyzation is also known. Nevertheless, the use of acombination of enzymes increases method costs.

Therefore, it would also be desirable to have a method which allowsobtaining suitable eggshell membrane solubilization efficacy with asingle proteinase such that it allows performing precise analysis of theeggshell membrane constituents, maintaining a high integrity of theproteins and of the rest of the metabolites that are present with aminimum method cost.

SUMMARY OF THE INVENTION

To solve the problems of the prior art, the present invention disclosesaccording to a first aspect a method for hydrolyzing eggshell membrane,comprising the step of treating a suitable amount of eggshell membranein a solution containing a denaturing agent, a reducing agent, a buffer,and an enzyme.

According to a second aspect, the present invention also discloses acomposition for hydrolyzing eggshell membrane suitable for use in themethod of the first aspect of the present invention. Specifically, thehydrolyzation composition comprises a denaturing agent, a reducingagent, a buffer, and an enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood in reference to thefollowing drawings illustrating a preferred embodiment of the invention,provided by way of example, which must not be interpreted as limitingthe invention in any way:

FIG. 1 is a graph showing the cumulative percentage of OVOMET solutionas a function of time for different dilution ratios.

FIG. 2 is a graph showing the % of absorption in human beings as afunction of logarithm of apparent permeability.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As mentioned above, the present invention discloses a method forhydrolyzing eggshell membrane as well as a composition for use in saidmethod. Given that eggshell membrane is extremely resistant todegradation and solubilization explained above, the method according tothe present invention resorts to the use of additives which can denaturethe proteins such that the enzymes can more readily access the bonds onwhich they perform their action.

Therefore, the method according to the preferred embodiment of thepresent invention comprises treating a suitable amount of eggshellmembrane in a solution containing a denaturing agent, a reducing agent,a buffer, and an enzyme, such that the main biological components of themembrane in the resulting product can be quantified without any type ofinterference, and the membrane hydrolysates thus obtained can then beused as raw materials in the preparation of food supplements,dermocosmetic products, and biopharmaceutical products, among others.

The preferred embodiments of the present invention include one or moreof the following characteristics:

-   -   the denaturing agent has detergent properties and an amphiphilic        nature, and is preferably an anionic surfactant, more        specifically sodium lauryl sulfate (SDS) or sodium taurocholate;    -   the reducing agent has a sulfurated nature, can break disulfide        bridges, will not generate interferences in subsequent        quantifications, and is selected from the group consisting of        food additives, more specifically sodium hydroxymethanesulfinate        (HOCH₂SO₂Na), sodium metabisulfite (Na₂S₂O₅), and dithiothreitol        (DTT), more preferably it is selected from sodium metabisulfite        and DTT, and even more preferably it is sodium metabisulfite;    -   the enzyme is a protease of plant origin with endopeptidase        activity from the group of cysteine proteases, preferably        selected from the group consisting of papain and bromelain.

To achieve proper eggshell membrane hydrolyzation, the quaternary,tertiary, and secondary structures of the eggshell membrane must firstbe altered so that the enzymes can finally access said membrane andhydrolyze it. To that end, the purpose of using a denaturing agent is tobreak the two- and three-dimensional structure of the proteins throughthe addition of a negative charge to the amino acids. In this manner, asthe proteins repel one another, their entanglement comes undone andaccess of the enzyme for hydrolysis becomes easier.

During the experiments, denaturing agents without detergent properties,such as urea, were tested without obtaining satisfactory dissolutionresults. However, when testing denaturing agents with detergentproperties, such as SDS and taurocholate, excellent results weresurprisingly obtained. Without wishing to be bound to any theory, it isbelieved that the structure of these detergents, which has a polarportion and another non-polar portion (amphiphilic nature), causesnon-polar products to be integrated in the aqueous solution.

Due to the large number of disulfide bridges existing in the proteins ofeggshell membrane, it was observed that in addition to using adenaturing agent it was necessary to use a reducing agent capable ofbreaking said disulfide bridges which caused some quaternary structuresto remain unaltered after the use of the denaturing agent. To that end,several reducing agents were studied with surprising results beingobtained for dithiothreitol (DTT) and sodium metabisulfite (Na₂S₂O₅).

Without wishing to be bound to any specific theory, the following schemeshows a possible mode of action of the additives used (denaturing agentand reducing agent) on the protein structure:

It is observed in the scheme that following the action of the SDS ortaurocholate, the proteins are exposed to receive the attack of theenzyme. The reducing agent ends up leading the proteins to have adenatured structure. Once peptides are formed due to the action of theenzyme, the SDS or taurocholate maintains them in solution.

Once it was demonstrated that the enzymes, which are ultimatelyresponsible for product hydrolysis, could fully access the proteins ofthe eggshell membrane, different enzymes were selected for efficiencytesting. To that end, different endopeptidases were evaluated becausethe objective was to separate the peptide bonds from the center of theproteins. On the other hand, given the large amount of cysteine residuesin eggshell membrane, in addition to the fact that disulfide bridges areestablished through the sulfur groups of said amino acid, it wassuitable to select enzymes belonging to the family of cysteineproteases. The efficacy of different enzymes was tested, and it wasobserved that cysteine proteases were in fact the most suitable.

Therefore, the preferred embodiments of the present invention provide amethod as well as a composition for suitably hydrolyzing eggshellmembrane. Said method and composition for hydrolyzing eggshell membranemay have several uses in the art. For example, according to a preferredembodiment the method and the composition for hydrolyzing eggshellmembrane allow determining the constituents of interest of the membrane(GAG, collagen, etc.) and using them as quality parameters to be takeninto account for the purpose of comparing similar products on themarket.

In a preferred embodiment, the method of the present invention comprisesthe step of treating an amount of eggshell membrane in a range ofbetween 5 and 155 mg/ml, preferably between 20 and 150 mg/ml, and morepreferably 70 mg/ml, in a solution containing sodium metabisulfite in arange of between 50 and 150 mM, preferably 100 mM, 0.5-5% SDS in 25-50mM HEPES buffer, preferably 50 mM, adjusted to a pH between 6 and 7,preferably 6.2, a 1% papain solution in sodium chloride in a range of0.05 to 0.5 M, preferably 0.15 M, being added to said solution until thefinal papain concentration in the solution is 0.05-0.5%.

According to another preferred embodiment of the invention, the methodand the composition for hydrolyzing eggshell membrane allow obtaining asoluble eggshell membrane product for the purpose of being obtained onan industrial scale and being used in various consumption sectors, forexample in the sector of food supplements, cosmetics, biopharmaceuticalproducts, etc.

In another preferred embodiment, the composition of the presentinvention comprises sodium metabisulfite in a range of between 50 and150 mM, preferably 100 mM, 0.5-5% SDS in 25-50 mM HEPES buffer,preferably 50 mM, adjusted to a pH between 6 and 7, preferably 6.2, and0.05-0.5% papain.

Several preferred embodiments of the present invention will be describedin greater detail below by means of specific examples, without theinvention being limited to said examples in any way.

Example 1 Assessment of Different Reducing Agents

A solution for solubilizing eggshell membrane was prepared at a ratio of10 mg of eggshell membrane/ml of solution. Said solution comprised adenaturing agent, and more specifically a denaturing agent havingdetergent properties and an amphiphilic nature such as SDS, at aconcentration of 0.5-5%. The solution also comprised a reducing agent,for which solutions of the following reducing agents were prepared at aconcentration of 100 mM-1 M: sodium sulfite (Na₂SO₃), sodiumhydrosulfite (Na₂S₂O₄), sodium hydroxymethanesulfinate (HOCH₂SO₂Na),sodium metabisulfite (Na₂S₂O₅), and dithiothreitol (DTT); thesesolutions were tested successively. Finally, a suitable buffer was addedfor maintaining the pH depending on the enzyme that was going to beused, having a concentration of 25-50 mM. Once the pH was adjusted witha pH-meter to the optimum pH for each enzyme, the enzyme previouslyprepared according to the manufacturer's specification at aconcentration of 1% was added to the solution, such that the finalconcentration of the enzyme with respect to the total solution was0.05-0.5%. The same was performed with each of the enzymes used in thepresent example.

Once all the components were mixed and homogenized, said mixture wasincubated at the maximum temperature of activity of each enzyme. Thelevel of hydrolysis was assessed after 1, 12, and 24 hours of incubationand the results were compared against a control that is performed andonly differs from the test solutions by the absence of enzyme.

The following table shows the results corresponding to the differenttests performed in Example 1.

Denaturing Reducing % Time ENZYME Nature pH agent agent solubilized (h)Papain Cysteine 6.2 SDS Na₂SO₃ 0 24 protease endopeptidase BromelainCysteine 6.2 SDS Na₂SO₃ 0 24 protease endopeptidase Ficin Cysteine 6.2SDS Na₂SO₃ 0 24 protease endopeptidase Protease Cysteine 7 SDS Na₂SO₃ 024 K protease endopeptidase Pepsin Endopeptidase 4 SDS Na₂SO₃ 0 24Papain Cysteine 6.2 SDS Na₂S₂O₄ 0 24 protease endopeptidase BromelainCysteine 6.2 SDS Na₂S₂O₄ 0 24 protease endopeptidase Ficin Cysteine 6.2SDS Na₂S₂O₄ 0 24 protease endopeptidase Protease Cysteine 7 SDS Na₂S₂O₄0 24 K protease endopeptidase Pepsin Endopeptidase 4 SDS Na₂S₂O₄ 0 24Denaturing % Time ENZYME Nature pH agent HOCH₂SO₂Na solubilized (h)Papain Cysteine 6.2 SDS HOCH₂SO₂Na 100 12 protease endopeptidaseBromelain Cysteine 6.2 SDS HOCH₂SO₂Na 100 12 protease endopeptidaseFicin Cysteine 6.2 SDS HOCH₂SO₂Na 0 24 protease endopeptidase ProteaseCysteine 7 SDS HOCH₂SO₂Na 0 24 K protease endopeptidase PepsinEndopeptidase 4 SDS HOCH₂SO₂Na 0 24 Denaturing Reducing % Time ENZYMENature pH agent agent solubilized (h) Papain Cysteine 6.2 SDS Na₂S₂O₅100 1 protease endopeptidase Bromelain Cysteine 6.2 SDS Na₂S₂O₅ 100 1protease endopeptidase Ficin Cysteine 6.2 SDS Na₂S₂O₅ 50 24 proteaseendopeptidase Protease Cysteine 7 SDS Na₂S₂O₅ 80 24 K proteaseendopeptidase Pepsin Endopeptidase 4 SDS Na₂S₂O₅ 0 24 Papain Cysteine6.2 SDS DTT 100 1 protease endopeptidase Bromelain Cysteine 6.2 SDS DTT100 1 protease endopeptidase Ficin Cysteine 6.2 SDS DTT 50 24 proteaseendopeptidase Protease Cysteine 7 SDS DTT 80 24 K protease endopeptidasePepsin Endopeptidase 4 SDS DTT 0 24

Therefore, it can be concluded based on the preceding results that, onlythree (sodium hydroxymethanesulfinate, sodium metabisulfite, and DTT) ofthe different tested reducing agents would allow 100% hydrolysis of thetested enzymes under the described conditions, and more specificallysodium metabisulfite and DTT showed better results.

Collagen and GAGs were quantified with the hydrolysates obtained afterusing DTT and sodium metabisulfite. In the case of the hydrolysatesobtained with DTT, interferences preventing proper quantification wereobserved. Therefore, in embodiments in which accurate quantification ofthe various components of the eggshell membrane is to be performed, useof sodium metabisulfite as a reducing agent is preferred.

Example 2

The following example sought to verify the optimum concentration ofsodium metabisulfite (Na₂S₂O₅) that will be used, for which papain wasused as an enzyme and different concentrations of Na₂S₂O₅ were used.Furthermore, whether or not there is a need to use the denaturing agent,SDS, was assessed, so use of said SDS was omitted when the maximumconcentration of Na₂S₂O₅ was used, a complete absence of hydrolysisbeing obtained, as demonstrated in the table below.

Specifically, a solution for solubilizing 10 mg of eggshell membrane/mlof solution was prepared. Said solution only contained 100 mM sodiummetabisulfite (Na₂S₂O₅) (1). Three other similar solutions were preparedwith an increasing order of concentration of Na₂S₂O₅, i.e., 5 mM (2), 50mM (3), and 100 mM (4), but in this case they contained 0.5-5% SDS in 50mM HEPES buffer adjusted to pH 6.2 with a pH-meter. It was verified thatthe optimum range of sodium metabisulfite (Na₂S₂O₅) for the presentinvention is between 50 and 150 mM. Papain previously activated in a0.15 M sodium chloride solution was added such that the finalconcentration of the enzyme in the mixture was 0.05-0.5%. Once all thecomponents were mixed and homogenized, said mixture was incubated at 38°C. for 12 h. The level of hydrolysis was evaluated after 12 hours ofincubation and the results were compared against a control that isperformed and only differs from the test solutions by the absence ofenzyme. A solution similar to the control was obtained for samples 1 and2, a completely clear, amber-colored solution demonstrating the completedisappearance of the eggshell membrane to be dissolved was obtained forsolution 4, and an intermediate result was obtained for solution 3.

The obtained results are shown in the following table.

(1) (2) SDS + (3) SDS + (4) SDS + Na₂S₂O₅ Na₂S₂O₅ Na₂S₂O₅ Na₂S₂O₅ Papain100 mM 5 mM 50 mM 100 mM % 0 0 70 100 solubilized

Example 3

In order to optimize the amount of membrane that could be hydrolyzedwith a papain concentration of 0.1%, different dilutions of the membraneto be hydrolyzed were prepared, where the best results were obtained fora membrane concentration between 20 and 150 mg/ml (although theinvention also works in the range of between 5 and 155 mg/ml), and morespecifically 70 mg/ml, as can be seen in the table below.

Specifically, different solutions with different amounts of eggshellmembrane were prepared such that final concentrations with respect tothe total solution volume of 20, 30, 40, 50, 70, 100, and 150 mg ofeggshell membrane/ml of solution were obtained. The solution contained100 mM sodium metabisulfite (Na₂S₂O₅), 0.5-5% SDS in 50 mM HEPES bufferadjusted to pH 6.2 with a pH-meter. 1% papain previously activated in a0.15 M sodium chloride solution was then added such that theconcentration of the enzyme with respect to the final volume of themixture was 0.05-0.5%. Once all the components were mixed andhomogenized, said mixture was incubated at 38° C. for 12 h and theresults were compared against a control that is performed and onlydiffers from the test solutions by the absence of enzyme. A clear,amber-colored solution was obtained, the color of which intensifies asthe membrane concentration increases, in addition to the presence of aprecipitate increasing, which indicates a lower level of hydrolysis.

Membrane, Papain, Time, % of mg/ml % h hydrolyzation 20 0.1 12 100 300.1 12 100 50 0.1 12 100 70 0.1 12 100 100 0.1 12 90 150 0.1 12 80

Example 4 Determination of Glycosaminoglycans (GAGs) in an Egg Membrane

GAGs are one of the components that are found in one of the highestproportions in egg membrane. The amount of beneficial effects that GAGscan provide to the human body following their intake has been describedon countless occasions; it is therefore of vital importance to provide atechnique which allows obtaining reliable and repetitive results. One ofthe methods most widely used for quantifying the GAG content of organicmaterial and providing some of the best results is the carbazole methoddescribed in detail in the Royal Spanish Pharmacopoeia (Real FarmacopeaEspañola), 2^(nd) edition, January 2002, 1472. One of the constraints ofthis method is that the samples to be quantified must be water-soluble.Given the insolubility of egg membrane, the primary requirement was toachieve solubilization of the egg membrane in water.

In order to validate the method and before quantifying the hydrolysates,different concentrations (0.02, 0.04, 0.06, and 0.08 mg/ml) of a knownGAG, i.e., hyaluronic acid (HA), were used. The results thus obtainedshowed that the carbazole method actually allowed reliably quantifyingthe presence of a GAG such as HA in a solution given that thecoefficient of correlation obtained was r²=0.9937).

Once the carbazole technique was validated, in order to be able tomeasure GAGs in the hydrolyzed egg membrane, GAGs in hydrolysates whichwere obtained with better results in Example 1 described above,specifically those which were obtained using sodium metabisulfite (A)and DTT (B) as a reducing agent, were quantified.

The same was performed with both hydrolysates as described below.

The following reagents were first prepared: a solution of 0.95 g ofsodium tetraborate in 100 ml of concentrated sulfuric acid, a solutionof 0.125 g of carbazole in 100 ml of anhydrous ethanol, and finally astock solution of D-glucuronic acid in 100 ml of water.

Once the required reagents as described above were prepared, 100 μl ofeach of hydrolysates A and B were diluted in 1000 μl of water. The testtubes were placed in an ice bath and 1.0 ml of test dilutions A and Bwas added. 5 ml of the previously prepared sodium tetraborate solutionkept in the ice bath were added to each tube. Once the test tubes werehermetically closed with glass stoppers, the content was stirred, andthe tubes were placed in a thermostatted bath at 90° C. for 10 minutes.The tubes were then cooled in an ice bath and 200 μl of a previouslyprepared alcoholic solution of carbazole were finally added to eachtube. The tubes were again placed in the bath at 90° C. for 15 minutesafter they were stoppered again and stirred, and the absorbance of testsolutions A and B at 530 nm was measured after cooling at roomtemperature. Along with the test samples, the same method was performedwith 4 samples with D-glucuronic acid concentrations of 6.5, 20, 40, and65 μg/ml obtained from the stock dilution to enable plotting a standardstraight line.

The standard samples and samples obtained from test sample A showed apink color similar to the color previously obtained when performing thetechnique validation test using HA.

However, test sample B showed a brown color in which it was impossibleto obtain absorbance results. Given that the only difference of bothtest samples A and B was the presence of a different reducing agent, itwas concluded that DTT caused interferences with the reagents of thecarbazole technique, and it was therefore concluded that the onlyreducing agent which can be used for hydrolyzing the membrane forsubsequent GAG quantification was sodium metabisulfite.

The technique thus used provided a GAG result of 5.08±0.4784 μg ofglucuronic acid/mg of Ovomet (egg membrane obtained by Eggnovo S.L.).

Example 5 Determination of Collagen in an Egg Membrane

Collagen is one of the major components of eggshell membrane. The levelsof collagen in egg membrane are determined in many published papers;however, the different results show fluctuations which to a certainextent are due to the different quantification techniques used. This isdue to the fact that all these techniques require prior membranesolubilization for subsequent analysis; therefore the level ofhydrolyzation of the membrane for solubilization is a vital parameter sothat the collagen values that are obtained are representative ofreality. There are now colorimetric quantification methods, such as theSircol® method which, despite having the advantage of being carried outrapidly, always has a lower precision compared to the precision of amethod based on HPLC determinations.

The proposed collagen quantification method is based on thehydroxyproline (Hyp) quantification method according to Hutson et al.2003 (J. Chromatogr. B 791: 427-430), with some modifications. Theresults obtained previously by Yu-Hong Zhao and Yu-Jiechi (Biotechnology8 (2):254-258 (2009), Characterization of Collagen from EggshellMembrane), demonstrating that the collagen of egg membrane is a type Icollagen, a decision is made to use type I collagen as a standard forthe determination. Furthermore, according to Dziewiatkowski D. et al,1972. (“Epimerization of trans-4-hydroxy-L-proline tocis-4-hydroxy-D-proline during acid hydrolysis of collagen”) it is knownthat two hydroxyproline isomers, i.e., L- and D-Hyp, are obtained duringcollagen degradation.

Based on the foregoing and before validating the method, L-Hyp, D-Hyp,and proline standards from Sigma-Aldrich were injected in the HPLC,these standards being prepared in a matrix at different concentrationsto enable identifying peaks corresponding to each of the isomers andproline expected to be obtained.

Once the characteristic peaks of the two hydroxyproline isomers and ofproline were determined and before quantifying the hydrolysates, aseries of solutions were prepared with different concentrations of mousetail type I collagen acquired from Sigma-Aldrich, collagen degradationchromatographic profile being obtained.

Given that the combination of the hydrolysis solution consisting of SDS,sodium metabisulfite, and papain was the one that had shown betterresults with the tests conducted up until now, a solution thusconstituted and with a concentration of the membrane to be dissolved of10 mg/ml was used.

100 μl of the hydrolysate thus obtained were then taken and 500 μl of 6N HCl were added, 400 μl were taken after homogenization, and another3600 μl of 6 N HCl were added. 100 μl of 2 mM sarcosine were addedthereto and it was kept in an oven at 110° C. for 18 hours in tubes thatare closed with their cover. Once cooled, it was neutralized with 6 NNaOH, bringing it to a pH of 9.5. 900 μl were taken from this solutionand 200 μl of 0.7 M borate buffer, pH 9.5, 100 μl of o-phthalaldehyde(OPA) solution, 100 μl of iodoacetamide reagent, and 300 μl of9-fluorenylmethyl chloroformate reagent (Fmoc-HCl) were added thereto,stirring with vortex for 10 seconds and leaving to stand for 1 minuteafter adding each of the solutions. Finally, it was washed three timeswith 2 ml of diethyl ether, stirring for 30 seconds in each case, twoclearly differentiated phases being obtained in each tube, an upperphase which is the organic phase and a lower phase which is the aqueousphase. The organic phase was discarded, and part of the aqueous phasewas taken and injected in the HPLC.

The chromatographic conditions were as follows: 5 μm Xterra® C18 columnhaving the dimensions of 25×0.46 mm. The phases used were A: 3% acetatebuffer, pH 4.3, and B: acetonitrile; in an isocratic ratio of 65/35,respectively, at a flow rate of 1 ml/min, and with a column temperatureof 25° C.

The chromatograms were optimized after 20 minutes using a fluorescencedetector at an excitation wavelength of 265 nm and without an emissionfilter.

The results thus obtained showed 35.31±4.8% of collagen present inOvomet (egg membrane obtained by Eggnovo S.L.).

For example, the following results are known in the prior art:

-   -   U.S. Pat. No. 6,176,376 B1-10% collagen    -   Yu-hong Zhao et al. 2009. Characterization of collagen from        eggshell membrane. Biotechnology, 8: 254-258-10% collagen.

The present invention shows better results than those observed in theliterature for egg membrane, which demonstrates that, taking intoaccount that all the results are obtained from membrane hydrolysates,the hydrolyzation of the membrane obtained by the method proposed inthis patent is superior to that described in other publications.

Example 6 In Vitro Studies of Cell Viability and Hydrolysate Absorption

Once the main biological components present in Ovomet were quantified,an in vitro study was designed to enable confirming, on one hand, thenon-toxicity of the hydrolysates obtained from the technique describedherein, and on the other hand, the absorption rate of said hydrolysatesin human beings.

To enable conducting both studies, a Caco-2 cell model forming amonolayer after a maturation process was used. To that end, the studywas conducted as described in detail below:

Cells were seeded at a density of 3×10³ per insert of 24 mm² and area of4.67 cm² in porous polyester (PET) membrane filters (0.4 μm) having 6wells (Transwell 3450; Costar, Corning). These inserts have twoseparated compartments, an apical compartment and another basalcompartment. The cells were seeded in each apical compartment dissolvedin a volume of 1.5 ml of culture medium. 2.5 ml of culture medium werethen added to the basal compartment.

For the cells to grow in the inserts, fresh culture medium was addedthereto every two days until 21 days lapsed so that cell growth wassuitable for the purpose of achieving the proper formation andmaturation of the multiple cell layers.

Once 21-day period following cell seeding ended, the cell monolayerswere checked in order to determine if they had intercellular gapsallowing the passage of any substance between the apical part and thebasal part, in order to assure that the passage of a substance to betested only occurred exclusively through the cells and not through anygap that may exist between them. To that end, transepithelial resistance(TER) was measured using a Millicell ohmmeter (ERS model, MilliporeCorp., Billerica Mass.; USA). Only those cell monolayers with resistancedata between 500 and 800 Ω·cm² were taken as suitable.

Once the 21-day period ended and the state of the cell monolayers wasconfirmed to be correct, the culture medium was removed and the cellswere bathed in a physiological solution referred to as HBSS (Hank'sbalanced salt solution) supplemented with 25 mM HEPES, pH 7.4 (Kis O etal., Pharm Res (2013) 30:1050-1064). The hydrolysate to be tested wasproduced as described below: 50 mg/ml of OVOMET dissolved in 50 mMHEPES, 1% SDS, and 100 mM sodium metabisulfite, and 0.1% bromelain for12 hours, under the conditions described above in Example 1.

Cell Viability Analysis by Means of Trypan Blue

Once the hydrolysate was obtained, different serial dilutions of thehydrolysate in HBSS (1:200, 1:400, 1:600, 1:800, 1:1000), which wereincubated with the cell culture at 37° C. and at 5% CO₂ for 4 hours,were made. After this time period elapsed, the culture medium wasremoved, and 400 μl of trypsin subsequently neutralized with 600 μl ofPBS and 2.5% fetal bovine serum were added. After successive washing,the cells were transferred to Eppendorf tubes which were centrifuged at5000 rpm for 5 minutes. The supernatant was removed and the precipitatewas resuspended in 300 μl of PBS and fetal bovine serum at the sameconcentrations as in the case described above.

To analyze cell viability, 50 μl of sample were taken and mixed with 50μl of 4% trypan blue. The samples were loaded in a Bürker chamber,observed under a light microscope, and the cells of 20 fields per samplewere counted, distinguishing living cells from dead cells.

The following selection criteria were established to select the optimumdilutions that showed better cell viability rates:

-   -   a—The cells do not detach once they are contacted with the        dilutions.    -   b—The count of dead cells in the Bürker chamber does not exceed        10%.    -   c—The cells observed under microscope have a suitable shape        (rounded with smooth edges).

It was observed that viability was greater than 90% in 1:400, 1:600,1:800, and 1:1000 dilutions, with 1:400, 1:600, and 1:800 dilutionsbeing chosen for the study.

Transport and Absorption Study

Once Caco-2 cell monolayers, the proper preparation of which had beentested as described above, were available, the cells were pre-incubated.To that end, a transport solution was added to both sides (both theapical side and the basal side) of the cell culture for 1 hour at 37° C.and 5% CO₂. Once pre-incubation ended, the transport solution wasremoved and the solution containing the membrane hydrolysate atdifferent dilutions (1:400, 1:600, and 1:800) was added to the apicalcompartment for incubation at 37° C. and with 5% CO₂, for 90, 120, 150,and 180 minutes. At the last point, a sample was further taken from theapical compartment. Hydroxyproline (Hyp) concentration was determined bymeans of HPLC on the basal side as an indicator of the intestinaltransport of Ovomet hydrolysate.

The following table shows the results of the transport study. Said tablehas the ratio corresponding to the chromatographic areas of Hyp obtainedin the different dilutions and at different times. The underlined valuesare Hyp values corresponding to the transport solution with differentdilutions of Ovomet hydrolysate.

Time, min. Solution OVOMET, 1/400 0.33406326  90 0.02141620 1200.1265950 150 0.04080866 180 0.03207032 OVOMET Solution, 1/6000.33406326  90 NQ 120 0.03725867 150 0.02343651 180 0.01011769 SolutionOVOMET, 1/800 0.20823490  90 0.02665078 120 0.01648812 150 0.01379862180 0.00788849

With these results, the cumulative percentage was calculated consideringthe values relative to the different hydrolysate concentrations.

Dilution of Ovomet hydrolysate 1/400 1/600 1/800 Cumulative % ofhydrolyzed Ovomet solution Time 100% 65.40% 50%  90  6.41 NQ  6.39 12010.20 11.52 10.35 150 22.41 18.77 13.67 180 32.01 21.90 15.56

With these values, linear regressions were plotted based on thecumulative percentage of OVOMET solution, where lines with anincreasingly greater slope are observed the higher the concentration ofthe tested hydrolysate solution is (see FIG. 1).

Once this data was known, the parameter referred to as “coefficient ofapparent permeability” (P_(app)) must be known given that there areseveral models linking the logarithm of P_(app) with absorption in humanbeings. The following equation was used to calculate P_(app):P_(app)=Q/A×Ci, where Q is the slope of the regression lines previouslycalculated (cumulative amount over time), A is the Transwell wellsection, and Ci is the concentration on the apical side. The data thuscalculated is shown in the following table.

Dilution P_(app) ( CM/ S ) Log P_(app) 1/400 1.606 E⁻⁰⁵ −4.79 1/6001.604 E⁻⁰⁵ −4.79 1/800 7.495 E⁻⁰⁶ −5.12

After knowing the Log P_(app) values as described above, they can becorrelated with the fraction absorbed in human beings after oraladministration expressed as a % of the dose of the administered productobtained in 4 different laboratories, see FIG. 2.

The following absorption results were obtained depending on thedifferent models mentioned.

Model % of absorption Artursson et al. 100 Cogburn et al.  95-100 Rubaset al.  93-100 Stewart et al. 45-65

The mentioned models are disclosed in the following sources:

Artursson et al. 1991. Correlation between oral drug absorption inhumans and apparent drug permeability coefficients in human intestinalepithelial (caco-2) cells. Biochemical and Biophysical ResearchCommunications 175 (3): 880-5.

Coburn et al. (1991). A model of human small intestinal absorptivecells. A. Transport barrier. Pharmaceutical Research 8 (2): 210-6.

Rubas et al. (1996). Flux measurements across Caco-2 monolayers maypredict transport in human large intestinal tissue. Journal ofPharmaceutical Science 85 (2):165-9.

Stewart et al. (1995). Comparison of intestinal permeabilitiesdetermined in multiple in vitro and in situ models: relationship toabsorption in humans. Pharmaceutical Research 12(5):693-9.

Although the present invention has been described above in reference tospecific preferred embodiments, one skilled in the art will understandthat various modifications and variations can be applied withoutdeparting from the scope of protection defined by the attached claims.Specifically, depending on specific application limits (costs, yieldsought, etc.) as well as the purposes of the application (obtainingcomponents that are useful in industry, quantifying componentsconstituting the membrane, etc.), different reducing agents, denaturingagents, buffers, and enzymes, as well as different amounts thereof, maybe used.

1-20. (canceled)
 21. A method for hydrolyzing eggshell membrane, comprising the step of treating a suitable amount of eggshell membrane in a solution containing a denaturing agent, a reducing agent, a buffer, and an enzyme, wherein the denaturing agent is sodium lauryl sulfate (SDS), the reducing agent is selected from the group consisting of sodium hydroxymethanesulfinate, sodium metabisulfite, and DTT, and the enzyme is an endopeptidase selected from the group of cysteine proteases.
 22. The method according to claim 21, characterized in that the reducing agent is selected from the group consisting of sodium metabisulfite and DTT.
 23. The method according to claim 22, characterized in that the reducing agent is sodium metabisulfite.
 24. The method according to claim 21, characterized in that the endopeptidase is selected from the group consisting of papain and bromelain.
 25. The method according to claim 21, characterized in that it comprises the step of treating an amount of eggshell membrane in a range of between 5 and 155 mg/ml, preferably between 20 and 150 mg/ml, and more preferably 70 mg/ml in a solution containing sodium metabisulfite in a range of between 50 and 150 mM, preferably 100 mM, 0.5-5% SDS in 25-50 mM HEPES buffer, preferably 50 mM, adjusted to a pH between 6 and 7, preferably 6.2, a 1% papain solution in sodium chloride in a range of 0.05 to 0.5 M, preferably 0.15 M, being added to said solution until the final papain concentration in the solution is 0.05-0.5%.
 26. A composition for hydrolyzing eggshell membrane comprising a denaturing agent, a reducing agent, a buffer, and an enzyme, wherein the denaturing agent is sodium lauryl sulfate (SDS), the reducing agent is selected from the group consisting of sodium hydroxymethanesulfinate, sodium metabisulfite, and DTT, and the enzyme is an endopeptidase selected from the group of cysteine proteases.
 27. The composition according to claim 26, characterized in that the reducing agent is selected from the group consisting of sodium metabisulfite and DTT.
 28. The composition according to claim 27, characterized in that the reducing agent is sodium metabisulfite.
 29. The composition according to claim 26, characterized in that the endopeptidase is selected from the group consisting of papain and bromelain.
 30. The composition according to claim 26, characterized in that it comprises sodium metabisulfite in a range of between 50 and 150 mM, preferably 100 mM, 0.5-5% SDS in 25-50 mM HEPES buffer, preferably 50 mM, adjusted to a pH between 6 and 7, preferably 6.2, and 0.05-0.5% papain. 